The restorative effects of platelet rich plasma and stem cells-derived exosomes on the genes involved in spermatogenesis of non-obstructive azoospermia Rat models
The restorative effects of platelet rich plasma and stem cells-derived exosomes on the genes involved in spermatogenesis of non-obstructive azoospermia Rat models
Azar Sheikholeslami,1Faezeh Davoodi,2Leila Naserpoor,3Mohsen Sheykhhasan,4Hoda Fazaeli,5,*
1. Department of Mesenchymal Stem Cells. Academic Center for Education, culture and Research (ACECR). Qom branch. Qom. Iran 2. Department of Mesenchymal Stem Cells. Academic Center for Education, culture and Research (ACECR). Qom branch. Qom. Iran 3. Department of Reproductive Biology. Academic Center for Education, culture and Research (ACECR). Qom branch. Qom. Iran 4. Department of Mesenchymal Stem Cells. Academic Center for Education, culture and Research (ACECR). Qom branch. Qom. Iran 5. Department of Mesenchymal Stem Cells. Academic Center for Education, culture and Research (ACECR). Qom branch. Qom. Iran
Introduction: A significant portion of male infertility is caused by azoospermia, which comes in two types: obstructive (OA) and non-obstructive (NOA). Adoption or the use of donated sperm are the two choices available to NOA patients who do not respond to medical therapy and are unable to conceive on their own. These patients still have a chance to become pregnant despite significant abnormalities in spermatogenesis. The only ways to conceive biological offspring in NOA patients are through testicular biopsy and assisted reproductive techniques since the impairment to spermatogenesis in these people is irreversible. In patients with NOA, these techniques have a limited rate of success, though. Furthermore, these relatively expensive and unavailable for azoospermic spermatogenesis failure technologies may use genetically impaired sperm in fertilization through natural barrier crossing techniques. However, despite the promising survival rates for children with cancer who may have undergone specific radiotherapy and chemotherapy, when they reach reproductive age, their fertility is impaired. As a result, the current target is to improve either their ability to produce sperm in their ejaculate or the probability that sperm can be successfully retrieved from the testis for ICSI. In this way, scientists are currently working to create azoospermia treatments based on stem cell transplantation. Extracellular vesicles (EVs), such as exosomes derived from mesenchymal stem cells (MSCs), have been found to have biological properties similar to those of stem cells while also providing important advantages over the cells from which they were made in the field of regenerative medicine. Unlike transplanted cells that cannot be recovered, EVs can get around most of the safety concerns related to direct cell transplantation. Additionally, unlike treatments involving transplanted cells, EVs are temporary and can be terminated quickly if negative effects appear. One of the most significant blood derivatives is platelet-rich plasma (PRP). More than 20 growth factors and other protein molecules, such as binding molecules and chemokines, are present in this bioactive substance, and they are involved in activities including cell proliferation, differentiation, and regeneration. These biological components and elements have given PRP therapeutic promise.
Methods: In this study which was conducted on 30 male Wistar rats 8-12 weeks old, we compared the effects of human PRP and exosomes released by adipose tissue-derived MSCs (AD-MSCs) on the restoration of in vivo spermatogenesis (Intra testis injection in NOA rat models). In the cell culture laboratory, AD-MSCs were isolated and cultured up to passage 3. After that, the exosomes were separated from their conditioned media. Peripheral blood from volunteer donors was obtained, and PRP was isolated in accordance with the manufacturer's instructions. In order to induce NOA in rats, they were injected with two doses of busulfan (10 mg/kg body weight intraperitoneally) at a 21-day interval. The rats received the following treatment at the time points of three days and two weeks: intratesticular injection of 100 microliters of exosome (500 mg/mL), 100 microliters of PRP, and 100 microliters of PBS in the AD-Exo, PRP, and sham groups, respectively. The rats were euthanized for future investigations two months after their last treatment.
Results: Based on the results of analyzing the expression of genes in various experimental groups, it was found that, with the exception of the SCP3 gene, which is involved in the late differentiation of spermatogonial cells, the expression of the other studied genes was significantly lower in the NOA and SHAM groups than in the control group (p≤0.05), indicating that NOA had been successfully induced, while significantly increased expression was reported in the treatment groups with AD-Exo and PRP (p≤0.05). Therefore, it appears that these therapies were successful in restoring the expression of spermatogenic genes including DAZL, DDX4, Miwi, and meiotic genes like stra8 and cyclin A1.
Conclusion: The ameliorating effects of PRP and exosomes secreted from AD-MSCs on the genes involved in spermatogenesis were confirmed in this study. It is obvious that additional research in histological, anatomical, plasma seminal antioxidant enzymes, the protein level of the genes studied in this study, etc. are required to determine the therapeutic impact of these treatments on patients with NOA.